Enhanced production of A|342is believed to be the pathogenic molecular alteration in PS1-linked autosomal dominant FAD. The overall goal of the studies proposed in this application is to examine APP-y-secretase interactions, in order to better understand the regulation of A|342/4o production by y-secretase. The underlying hypothesis is that y-secretase, a complex of four membrane associated proteins, can adopt different conformations which differentially favor Ap40 or Ap42 production. We postulate that these conformations become preferred either through structural changes in core y-components or allosteric modifications of the complex. For example, using FRET based assay we have demonstrated that FAD associated PS1 mutations that increase A(342 (Berezovska et al., 2005) lead to a specific alteration in PS1 conformation. Iwatsubo and colleagues recently demonstrated that extension of the N terminus of Pen-2 (Isoo et al., 2007) similarly leads to structural change in y-complex and enhances the Ap42/40 ratio. Presumed allosteric modulators, such as some nonsteroidal anti-inflammatory drugs, which lower A|342/4o ratio (Lleo et al., 2004), or fenofibrate, which increases A|342/4o ratio, alter PS1 conformation in opposite directions. We therefore postulate that the conformational profile of the y- secretase complex measured by FRET reflects functional changes in the y-complex that lead to alterations in A(342/40 ratios. A second, related hypothesis is that the amount of Ap and A|342/40 ratio may also depend on how and where APP is presented to y-secretase. FAD mutations near the y-cleavage site in APP, and interactors that modify either APP or PS1 trafficking, and the cellular site of cleavage, may act in this fashion. The advanced imaging methodologies developed in these experiments, designed to monitor protein conformation and protein - protein interactions in living cells, are based on FRET, bimolecular complementation, and total internal reflection microscopy. Together with biochemical assays, sensitive ELISA, and interactions with other members of the PPG team, the proposed experiments will allow us to explore the effects of subcellular compartments and Pen2 alterations (Aim1), APP and substrate manipulations (Aim2), and pharmacological interventions (Aim3). Importantly, the development of these assays will allow us to examine the consequences on y-secretase of interacting molecules, mutations, and novel substrates as they emerge from the PPG effort.